The field of this invention is related to sample loading, stacking, and separation in a microfluidics device.
Microfluidics is revolutionizing the way activities are performed in a substantial proportion of chemical and physical operations. One area of microfluidics is the manipulation of small volumes of liquids or liquid compositions on a solid substrate, where a network of channels and reservoirs are present. By employing electric fields with electrically conducting liquids, volumes and/or ions can be moved from one site to another, different solutions formed by mixing liquids and/or ions, reactions performed, separations performed, and analyses carried out. In fact, in common parlance, the system has been referred to as xe2x80x9ca laboratory on a chip.xe2x80x9d Various prior art devices of this type include U.S. Pat. Nos. 6,010,608, 6,010,607, 6,001,229, 5,858,195, and 5,858,187 which are a family of applications concerned with injection of sample solutions. See also, U.S. Pat. No. 5,599,432, EPA 0620432, and Verheggen et al., J. of Chromatography 452 (1988) 615-622.
In many of the operations, there is an interest in electrophoretically separating multiple sample components contained in dilute samples, e.g., samples with concentrations of sample components in the femptomolar to nanomolar range. Efficient electrophoretic injection of dilute samples frequently results in large sample volumes and poor resolution of the sample components.
It would thus be desirable to provide an electrophoretic system for improved separation and resolution of sample components, particularly where the sample components are present at nanomolar concentrations or less. It would be further desirable to provide a method of adjusting separation conditions as to maximize electrophoretic separation and resolution.
In one aspect, the invention includes a microfluidics system for use in electrophoretic separation of components having a given negative or positive charge and contained in a dilute sample. The system includes a microfluidics device having a substrate and a channel network formed therein. The channel network has a separation channel and first and second side channels that intersect the separation channel at axially spaced positions therealong. The two side channels partition the separation microchannel, in an upstream to downstream direction, into an upstream channel region upstream of intersection with the first side channel, a sample-volume channel region between the intersections of the two side channels, and a downstream separation channel region downstream of the second side channel intersection. The ratio of the lengths of the sample-volume region to the separation channel is between about 1:50 to 1:1.
The channel network is designed to be loaded initially by filling the upstream channel region with a trailing-edge electrolyte, filling the sample-volume channel region with the dilute sample, and filling the separation channel region, with a leading-edge (LE) electrolyte.
Also included in the device are upstream and downstream reservoirs communicating with the upstream and downstream ends of the separation channel, respectively, and first and second reservoirs communicating with the first and second side channels, respectively, opposite the side channel intersections with the separation channel. Upstream and downstream electrodes in the system are adapted to contact liquid contained in the upstream and downstream reservoirs, respectively.
A control unit in the system includes a power source for applying a voltage potential across the upstream and downstream electrodes, under conditions such that, with the upstream channel region filled with a trailing-edge electrolyte, the sample-volume channel region filled with the dilute sample, and the separation channel region filled with a leading-edge electrolyte, the sample stacks into a relatively small sample volume before hydroxyl- or hydrogen-ion migration into and through the sample-volume region is effective to overtake the charged sample components, wherein continued application of an electric potential across the channel ends causes charged sample components in the stacked sample volume to separate by zone electrophoresis.
In one general embodiment, the upstream channel region is filled with a trailing-edge electrolyte containing a selected concentration of a titratable species. Application of the voltage potential is effective to cause charged components in the sample to stack by isotachophoresis, and, at the same time, electrolytic hydroxyl or hydrogen ions formed by electrolysis at the upstream-end electrode to migrate into the trailing-edge electrolyte, titrating the titratable species therein. The concentration of titratable species in the trailing-edge electrolyte is selected, in relation to the lengths of the upstream channel region and sample-loading volume, to permit the sample to stack into a relatively small sample volume before electrolytic-ion migration from the upstream electrode into and through the sample-volume region is effective to overtake the charged sample components, wherein continued application of an electric potential across the channel ends causes charged sample components in the stacked sample volume to separate by zone electrophoresis.
In another general embodiment, the upstream channel region includes a pair of upstream reservoirs, one containing the TE electrolyte, and the other containing a source of hydroxyl or hydrogen ions, e.g., a basic or acidic solution. The control unit is operated to initially apply a voltage potential across electrodes in contact with the one upstream reservoir and the downstream channel end, and subsequently, to apply a voltage potential across electrodes in contact with the other upstream reservoir and the downstream channel end.
The system may be used to detect charged sample components present at nanomolar concentrations or less, where the ratio of the lengths of the sample-volume region to the separation channel in the device is between about 1:50 to 1:1.
The system may include one of a plurality of different microfluidics devices having different channel-length ratios between 1:50 and 1:1. In this embodiment, the control unit is operable to calculate the approximate concentration of titratable species in the trailing-ion electrolyte required for any selected microfluidics device length ratio.
The control unit may also be operable to load (i) the downstream channel region with the leading-edge electrolyte, by applying an electrokinetic voltage across the downstream reservoir and one of the first and second reservoirs, (ii) the upstream channel region with the trailing-edge electrolyte, by applying an electrokinetic voltage across the upstream reservoir and one of the first and second reservoirs, and (iii) the sample volume region by applying a fluid-motive force effective to move sample contained in one of the first and second reservoirs through the sample-volume region and toward the other of the first and second reservoirs. The device in this embodiment may include first and second electrodes adapted to contact liquid contained in the first and second reservoirs, respectively, where the control unit is operable to load the sample volume region by applying an electrokinetic voltage across the first and second electrodes.
The control unit is preferably operable to apply having across the upstream and downstream electrodes, a voltage potential characterized by a constant current, a constant voltage or constant power.
In another aspect, the invention includes a method of separating components having a given negative or positive charge and contained in a dilute sample. Initially a separation microchannel having, in an upstream to downstream direction, an upstream channel region, a sample-volume channel region, and a downstream separation channel region, is loaded so as to fill the upstream channel region with a trailing-edge electrolyte containing a selected concentration of a titratable species, the sample-volume channel region, with the dilute sample, and the separation channel region, with a leading-edge electrolyte.
There is then created an electrical field potential across the channel, by applying a voltage potential across electrodes in contact with the upstream and downstream channel ends, initially causing charged components in the sample to stack by isotachophoresis, and subsequently causing hydroxyl or hydrogen ions to migrate into the trailing-edge electrolyte, titrating the titratable species therein, under conditions that that permit the sample to stack into a relatively small sample volume before hydroxyl- or hydrogen-ion migration into and through the sample-volume region is effective to overtake the charged sample components, wherein continued application of an electric potential across the channel ends causes charged sample components in the stacked sample volume to separate by zone electrophoresis.
In one general embodiment, the upstream channel region is filled with a trailing-edge electrolyte containing a selected concentration of a titratable species. Application of the voltage potential is effective to cause charged components in said sample to stack by isotachophoresis, and, at the same time, electrolytic hydroxyl or hydrogen ions formed by electrolysis at the upstream-end electrode to migrate into the trailing-edge electrolyte, titrating the titratable species therein. The concentration of titratable species in the trailing-edge electrolyte is selected, in relation to the lengths of the upstream channel region and sample-loading volume, to permit the sample to stack into a relatively small sample volume before electrolytic-ion migration from the upstream electrode into and through the sample-volume region is effective to overtake the charged sample components, wherein continued application of an electric potential across the channel ends causes charged sample components in the stacked sample volume to separate by zone electrophoresis.
The trailing-edge electrolyte preferably includes a trailing-edge ion and a titratable counter-ion buffer at said selected concentration. Where the electrolytic ions formed at the upstream-end electrode are hydroxyl ions, the titratable counter-ion buffer may be a TRIS buffer.
In another general embodiment, the upstream channel region includes a pair of upstream reservoirs, one containing the trailing-edge electrolyte, and the other containing a source of hydroxyl or hydrogen ions. Initially a voltage potential is applied across electrodes in contact with the one upstream reservoir and the downstream channel end, and subsequently, a voltage potential is applied across electrodes in contact with the other upstream reservoir and the downstream channel end.
For use in detecting charged sample components present at nanomolar concentrations or less, the ratio of sample volume before and after isotachophoretic stacking is at least about 10:1, and may be at least about 50:1. For in detecting charged sample components present at picomolar or less concentrations, the ratio of sample volume before and after isotachophoretic stacking may be at least about 100:1.
The ratio of the lengths of the sample-volume region to the separation channel is preferably between about 1:50 to 1:1, more preferably between about 1:10 to 1:2.
In one exemplary method, the leading-edge electrolyte contains a negatively charged leading-edge ion having an effective conductivity greater than that of the sample ions and a concentration between 1-50 mM, the trailing-edge electrolyte contains a negatively charged trailing-edge ion having an effective conductivity lower than that of the sample ions and a concentration of between 1-50 mM, and both electrolytes have a positively charged buffer at a selected concentration between about 2 and 50 mM.
For use in separating a plurality of electrophoretic tags contained in a sample, each tag may have a detectable moiety and a mobility modifier that confers on the tag, a unique electrophoretic mobility. The method further includes the steps, after separating the tags electrophoretically, of detecting the separated tags, and from their electrophoretic mobilities and concentration, obtaining information about a biomolecular interaction.
Where the tags are contained on branched polymer structures and linked thereto through photo-labile linkages, the method may further include the step, after permitting branched structures in the sample to stack into a relatively small sample volume, of irradiating the branched structures to release the tags therefrom. Continued application of an electric potential across the channel ends causes the tags in the stacked sample volume to separate by zone electrophoresis.
These and other objects of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.